﻿A new species of Lumbrineriopsis (Annelida, Eunicida, Lumbrineridae) from southeastern Brazil

﻿Abstract Lumbrineriopsisdulcissp. nov. is morphologically described from the continental shelf and slope of Espírito Santo and the Campos Basin of Rio de Janeiro state, southeastern Brazil, at depths between 14 and 400 m. Lumbrineriopsismucronata is the only species of the genus recorded until now in Brazil. The new species differs from other congeneric species in its jaw-apparatus morphology with unfused mandibles and a fixed number of simple limbate chaetae and simple, bidentate, hooded hooks in each parapodium. This paper aims to fill the gap in knowledge on the family Lumbrineridae, which has not been studied in Brazil for the last 25 years and provides the first record of the genus from Espírito Santo and Rio de Janeiro states. This record is significant given the damage to the marine ecosystem of the Espírito Santo region due to the 2015 rupture of the Samarco mining company dam, the largest environmental disaster in Brazil’s history. In addition, this region has important environmental conservation units such as Costa das Algas Environmental Protection Area, Santa Cruz Wildlife Refuge, and Comboios Biological Reserve. All these preserved areas are of paramount importance for the protection of marine biological diversity.


Introduction
The family Lumbrineridae Schmarda, 1861 is composed of species with similar external morphology. Their identification was based, until 1990, on characters of the prostomium and parapodial lobes, as well as the type of chaetae. Consequently, the classification resulted in a generic system that arranged the existing species into three or four genera (Hartman 1944;Fauchald 1970;Zanol et al. 2021), which lead to descriptions with insufficient information. In addition, jaw structure was little explored. Studies by Orensanz (1973Orensanz ( , 1990 proposed new morphological characters, especially related to jaw apparatus, representing an advancement of the taxonomy of the family and promoting the description of new genera and species (Orensanz 1990;Carrera-Parra 2001; Carrera-Parra and Orensanz 2002;Carrera-Parra 2004;Carrera-Parra 2006;Katsiaras et al. 2017).
Lumbrineridae comprises about 279 valid species in 19 genera (Carrera-Parra 2006; Zanol et al. 2021). In Brazil, 32 species have been recorded belonging to the genera Augeneria, Cenogenus, Lumbricalus, Lumbrinerides, Lumbrineriopsis, Lumbrineris, Lysarete, Ninoe, and Scoletoma; they occur from the intertidal zone to the continental shelf and slope, reaching 1126 m deep, on both consolidated and unconsolidated substrates, as well as associated with algae, sponges, and corals (Costa et al. 2017;Amaral et al. 2023). However, only Lana (1995a, 1995b) have carried out more comprehensive taxonomic studies of the family and described 17 species from southern and southeastern coasts of Brazil, including two new species, Scoletoma mainae and Scoletoma curtolobata.
The genus Lumbrineriopsis was erected by Orensanz (1973) for a group very small, narrow and long-bodied lumbrinerids having simple, bidentate, hooded hooks, long and slender maxillary carriers, and maxillae IV with a fringe of denticles. Two species were initially known: L. paradoxa (Saint-Joseph, 1888) and L. mucronata (Ehlers, 1908). Subsequently, L. tsushimaensis Imajima & Higuchi, 1975, L. gardineri Miura, 1980, and L. gasconiensis Miura, 1980 were described. Lumbrineriopsis mucronata has been recorded in Brazil where it occurs in silty-clayey and sandy-mud bottoms of bays, from 8 to 90 m deep, in the states of Rio de Janeiro and Santa Catarina (Camargo and Lana 1995a).
Herein, Lumbrineriopsis dulcis sp. nov. is described based on material from the states of Espírito Santo and Rio de Janeiro. The new species is grouped together with L. mucronata and L. paradoxa, whose distinctive feature is the presence of unfused jaws. This is the first record of the genus from Espírito Santo state, which is significant given the damage to the marine ecosystem in this region due to the rupture of the dam belonging to the Samarco mining company. In 2015 the company dumped 50 million m 3 of ore tailings into the Doce River, which flows into an estuary in the Atlantic Ocean. This was the biggest environmental disaster in the history of Brazil. In addition, this region has important environmental conservation units such as Costa das Algas Environmental Protection Area, Santa Cruz Wildlife Refuge, and Comboios Biological Reserve. All these preserved areas are of paramount importance for the protection of marine biological diversity.

Material and method
The specimens were collected along southeastern Brazil during two major Brazilian oceanographic research projects carried out between 2008 and 2013 in soft bottoms from 12 to 3301 m depth and coordinated by CENPES/PETROBRAS ("Assessment of the environmental heterogeneity of the Campos Basin" (HABITATS) and "Marine environmental characterization of the Espírito Santo and northern portion of the Campos Basins" (AMBES)). The collected samples were fixed in 4% formaldehyde, and the specimens were preserved in 70% alcohol.
For detailed visualization of the jaw apparatus, some individuals were immersed in 0.2% sodium hypochlorite for 12 min and subsequently immersed in Hoyer's solution. Chaetigers from anterior, median, and posterior regions, including parapodia, were dissected and immersed in Aquatex aqueous mounting medium. Individuals were examined using both Zeiss Stereo Discovery v. 2.0 and Zeiss Axioskop 2 Plus microscopes. Two specimens for scanning electron microscopy (SEM) imaging were dehydrated in a series of baths from 70% to 100% ethanol, then carried to the critical-point drying, metalized, and analyzed using a JEOL 6610 LV scanning electron microscope at the Laboratory of Electron Microscopy (LME), Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP). The specimens are deposited at the Polychaeta Collection (ZUEC-POL) of the Museu de Diversidade Biológica (MDBio), Instituto de Biologia, UNICAMP and the Polychaeta Collection (MZUSP) at the Museu de Zoologia, Universidade de São Paulo (USP), Brazil.
Based on the morphology of mandibles (Figs 7, 8A-C) the specimens were divided into two groups: up to 0.29 mm wide (juvenile specimens) and between 0.3 and 0.9 mm (adults).

Systematics Class Polychaeta Grube, 1850 Order Eunicida Dales, 1962 Family Lumbrineridae Schmarda, 1862
Genus Lumbrineriopsis Orensanz, 1973 Diagnosis. Antennae and eyes absent. Notopodia small, without branchiae. Limbate chaetae and simple bidentate hooded hooks. Pygidium without anal cirri. Jaw apparatus with four pairs of maxillae, maxillary carriers longer than MI, joined to 1/2 of its base. MI forceps-like, without inner accessory teeth, with attachment lamella. MII with ligament, wide attachment lamella along 2/3 of posterior lateral edge; without connecting plates. MIII completely pigmented, narrow attachment lamella along 1/2 of posterior lateral edge. MIV completely pigmented, wide attachment lamella. Mandibles unfused, fused along the entire inner margin or up to 3/4 of its length (modified from Carrera-Parra 2006 (14): 0.1-0.25 mm wide, 5-11 mm long, 11-54 chaetigers. Body yellow or whitish in preserved specimens. Prostomium long, acuminate, 1.5-2.5 times longer than peristomium (Figs 1A, B, 4). Peristomial rings slightly demarcated, both with equal length or first ring twice as long as the second one, both slightly narrower than the following segments (Figs 1A, B, 4, 5A); without mouth pads. Jaw apparatus light brown (Fig. 7). MI forceps-like, with attachment lamella, basal external projec-tion, basal inner portion sharper and connected to the maxillary carriers (Figs 1E, 7E). Maxillary carriers 1.5 to twice as long as MI, with tiny irregular structures in anterior end (Figs 1E,7E). MII connected to the inner base of MI by a ligament, 6 or 7 teeth with curved and rounded end, the 2 basal teeth being the smallest ( 2F). Pygidium rounded, without anal cirri (Fig. 5D). Etymology. The specific epithet "dulcis" is a tribute to the Doce River, which flows into an estuary in the Atlantic Ocean, where L. dulcis is recorded.    Distribution and habitat. The distribution of this new species encompasses the states of Espírito Santo and Rio de Janeiro in southeastern Brazil, from the mouth of the Doce River to the continental slope (at depths between 14 and 400 m) in mud, sand, mixed sandy-mud bottoms, in sand with biological debris, and between limestones.
Remarks. The nuchal organ was observed dorsally at the base of the prostomium in specimens analyzed under SEM (Fig. 4C) but is difficult to visualize under the stereoscopic microscope due to the small size of the specimens.
The specimens are very small, and the jaw apparatus very delicate, especially the mandibles (Figs 7A, B, E, F, 8A-C). There is no evidence of a chitinous lig- ament joining the pair of mandibles. In juveniles (Fig. 8A-C), each mandible is composed of a distal denticulated plate and a chitinous longitudinal rod, which remains prominent in the adult (on its inner side) and from where the mandible expands laterally and grows, which is the pattern observed in many other species of Eunicida. The color of this lateral portion can vary from intense light brown (Fig. 7A, B), colorless (Fig. 7D, E) to almost transparent (Fig. 7F), while the internal chitinous rod and anterior end remain always visible.
The notopodium, which is described as slightly developed in the description of the genus (Carrera-Parra 2006), was not observed in the new species. The post-chaetal lobe, which is digitiform, relatively short, and projected backward, sometimes is not properly positioned for visualization under the stereoscopic microscope, or sometimes appearing to be absent in slide preparation (Fig. 8F,  G). Thus, care is required in positioning and placing of the coverslip to prevent some structures from being overlooked during observation under the microscope. However, the post-chaetal lobe is clearly digitiform in SEM images (Fig. 5C). There is a fixed number of two limbate chaetae along the entire body.

Discussion
Of the five known species so far, two groups can be clearly observed: one group with unfused mandibles (L. paradoxa and L. mucronata) and another with fused  Although the mandibles are not fused, both are connected by a chitinous ligament (Imagima and Higuchi 1975). Traditionally, a higher taxonomic value is given to the set of maxillae, with the mandibles supporting the definition of the species. In this study, while the maxillae were highly considered, greater attention was paid to the pair of unfused mandibles, a rare characteristic among Lumbrineridae. Lumbrineriopsis dulcis sp. nov. differs markedly from L. tsushimaensis, L. gasconiensis, and L. gardineri, since these species have fused mandibles, with or without growth rings at the distal end. Thus, L. dulcis sp. nov. resembles L. paradoxa and L. mucronata (Table 1).
Although Miura (1980) deemed L. paradoxa and L. mucronata to be synonyms, the criteria adopted were few and the features superficial, while the original descriptions of Saint-Joseph (1888) and Ehlers (1908), have important information that was not considered by Miura (1980). Lumbrineriopsis paradoxa and L. mucronata differ from each other in the shape and number of teeth of MII, number of limbate chaetae along the body, and the presence of a denticulated plate in the distal portion of the mandibles of L. paradoxa, whereas in L. mucronata there is a cutting border with some larger teeth (Table 1).
Both species have been recorded in other localities (Fauvel 1914(Fauvel , 1923Orensanz 1973;Camargo and Lana 1995a) but described as having partially fused mandibles. However, Saint-Joseph (1888) described L. paradoxa (based on a single slide-mounted specimen) as having two thin, juxtaposed rods which enlarge at the anterior end. Ehlers (1908) described L. mucronata (also based on a single specimen) as having a distal plate supported by "thin rods" (plural), whose cutting edge has some large teeth. Although Ehlers (1908) explained that he analyzed the mandibles laterally, the drawing clearly shows one of the "thin rods", confirming the presence of unfused mandibles. Miura (1980) examined type material of both species, but also from localities in addition to the type localities, but he did not mention anything that resembled fused mandibles, a calcareous plate, or growth rings.
Regarding the stage of development of these two species, Saint-Joseph (1888) described a specimen 0.24 mm wide and with 93 setigers, which clearly is an adult individual, given the high number of setigers already added to the body. On the other hand, Ehlers (1908) described a complete specimen 0.5 mm wide and only 60 setigers, considered by him to be an apparent juvenile. Comparatively, it is observed that all species of the genus Lumbrineriopsis are very small, narrow, and long, ranging from 0.24 mm (L. paradoxa) (through 0.9 mm; L. dulcis) to 1 mm wide (L. tsushimaensis). Thus, although the presence of unfused mandibles is also a characteristic of young stages in Lumbrineridae, it is possible to affirm that Lumbrineriopsis clearly has species with fused or unfused mandibles.
Therefore, in view of this scenario, as well as the need for further clarify the validity of L. paradoxa and L. mucronata, we have followed Miura (1980) in understanding that the mandibles are not fused. However, we have considered both valid, based on the information from the original descriptions and the Worms Database (2023). Lumbrineriopsis dulcis differs from L. mucronata in the coloration of the jaw apparatus and number of teeth of MII, MIII, and MIV, in addition to the number of limbate chaetae per parapodium (Table 1). Lumbrineriopsis paradoxa, on the other hand, differs from L. dulcis in its body coloration and the shape of MII, which, in the former is elongated and, in the latter, rounded. Both species also differ in relation to the number of teeth of MII and MIV, number of limbate chaetae in the posterior region, and number of aciculae per parapodium (Table 1).
Taxonomic studies of the genus carried out in South America by Orensanz (1973) and Camargo and Lana (1995a) have recorded the occurrence of L. mucronata. However, in these two studies, the mandibles are almost completely fused, with growth rings in the anterior end, indicating that the specimens may belong to another species. Camargo and Lana (1995a) also mentioned the simple bidentate hook with a basal tooth forming a 90° angle with the distal tooth, while Orensanz (1973) mentioned a double basal external projection in MI. Such characters are not present in L. dulcis. Uebelacker (1984) described L. paradoxa with unfused mandibles from the Gulf of Mexico, with characteristics similar to L. dulcis and having the morphology of the juvenile mandibles, but differing from it by the absence of teeth in MIII and also bearing a greater number of limbate chaetae (2 or 3), hooks (1 or 2), and aciculae (2 or 3), in addition to the black coloration of the mandibles in juveniles, with several rows of teeth present in the anterior plate.
Considering the peculiar characteristics of Lumbrineriopsis, with such elongated maxillary carriers, multidentate MIV, and unfused mandibles in some species, a more in-depth analysis of its phylogenetic positioning within Lumbrineridae is necessary, since questions have already been raised (Saint Joseph 1888). Studies involving micro-CT are interesting to elucidate important details about jaw apparatus.